Hydroxylation of the C13(2) and C18 carbons of chlorophylls by heme and molecular oxygen

Following extraction from photosynthetic organisms, chlorophylls are prone to reactions including demetalation, dephytylation and specific oxidations of the exocyclic ring E, termed allomerizations. Allomerization of chlorophylls has been well-characterized in methanol and to a lesser extent in aqueous solution. Here we detail novel allomerization-like reactions of chlorophyll a and chlorophyll b. In the presence of heme, detergent-solubilized chlorophyll a is hydroxylated at its C13² position in ring E and, surprisingly, the C18 position in ring D. Two major oxidation products are synthesized - a C13²-OH and a C13²-OH, C18-OH derivative of chlorophyll a. We track the origin of the oxygen atoms added in these hydroxylated chlorophylls using ¹⁸O₂ labeling and demonstrate that the additional oxygen atoms are derived from molecular oxygen. A similar heme-catalyzed reaction is also observed using chlorophyll b as a substrate. These results highlight the need for care when dealing with extracted chlorophylls and demonstrate an unusual hydroxylation of the C18 position of chlorophylls in the presence of heme.